Method of regulating glucose metabolism, and reagents related thereto

ABSTRACT

The present invention provides methods and compositions for modification and regulation of glucose and lipid metabolism, generally to reduce insulin resistance, hyperglycemia, hyperinsulinemia, obesity, hyperlipidemia, hyperlipoprotein-emia (such as chylomicrons, VLDL and LDL), and to regulate body fat and more generally lipid stores, and, more generally, for the improvement of metabolism disorders, especially those associated with diabetes, obesity and/or atherosclerosis.

FUNDING

[0001] Work described herein was supported by finding from the NationalInstitutes of Health. The United States Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

[0002] Diabetes adversely affects the way the body uses sugars andstarches which, during digestion, are converted into glucose. Insulin, ahormone produced by the pancreas, makes the glucose available to thebody's cells for energy. In muscle, adipose (fat) and connectivetissues, insulin facilitates the entry of glucose into the cells by anaction on the cell membranes. The ingested glucose is normally convertedin the liver to CO₂ and H₂O (50%); to glycogen (5%); and to fat(30-40%), the latter being stored in fat depots. Fatty acids from theadipose tissues are circulated, returned to the liver for re-synthesisof triacylglycerol and metabolized to ketone bodies for utilization bythe tissues. The fatty acids are also metabolized by other organs. Fatformation is a major pathway for carbohydrate utilization.

[0003] The net effect of insulin is to promote the storage and use ofcarbohydrates, protein and fat. Insulin deficiency is a common andserious pathologic condition in man. In insulin-dependent (IDDM or TypeI) diabetes the pancreas produces little or no insulin, and insulin mustbe injected daily for the survival of the diabetic. Innoninsulin-dependent (NIDDM or Type II) diabetes the pancreas retainsthe ability to produce insulin and in fact may produce higher thannormal amounts of insulin, but the amount of insulin is relativelyinsufficient, or less than fully effective, due to cellular resistanceto insulin.

[0004] Diabetes mellitus (DM) is a major chronic illness found in humanswith many consequences. Some complications arising from long-standingdiabetes are blindness, kidney failure, and limb amputations.Insulin-dependent diabetes mellitus (IDDM) accounts for 10 to 15% of allcases of diabetes mellitus. The action of IDDM is to cause hyperglycemia(elevated blood glucose concentration) and a tendency towards diabeticketoacidosis (DKA). Currently treatment requires chronic administrationof insulin. Non-insulin dependent diabetes mellitus (NIDDM) is marked byhyperglycemia that is not linked with DKA. Sporadic or persistentincidence of hyperglycemia can be controlled by administering insulin.Uncontrolled hyperglycemia can damage the cells of the pancreas whichproduce insulin (the β-islet cells) and in the long term create greaterinsulin deficiencies. Currently, oral sulfonylureas and insulin are theonly two therapeutic agents available in the United States. fortreatment of Diabetes mellitus. Both agents have the potential forproducing hypoglycemia as a side effect, reducing the blood glucoseconcentration to dangerous levels. There is no generally applicable andconsistently effective means of maintaining an essentially normalfluctuation in glucose levels in DM. The resultant treatment attempts tominimize the risks of hypoglycemia while keeping the glucose levelsbelow a target value. The drug regimen is combined with control ofdietary intake of carbohydrates to keep glucose levels in control.

[0005] In either form of diabetes there are widespread abnormalities. Inmost NIDDM subjects, the fundamental defects to which the abnormalitiescan be traced are (1) a reduced entry of glucose into various“peripheral” tissues and (2) an increased liberation of glucose into thecirculation from the liver. There is therefore an extracellular glucoseexcess and an intracellular glucose deficiency. There is also a decreasein the entry of amino acids into muscle and an increase in lipolysis.Hyperlipoproteinemia is also a complication of diabetes. The cumulativeeffect of these diabetes-associated abnormalities is severe blood vesseland nerve damage.

[0006] Endocrine secretions of pancreatic islets are regulated bycomplex control mechanisms driven not only by blood-borne metabolitessuch as glucose, amino acids, and catecholamines, but also by localparacrine influences. Indeed, pancreatic α- and β-cells are criticallydependent on hormonal signals generating cyclic AMP (cAMP) as asynergistic messenger for nutrient-induced hormone release. The majorpancreatic islet hormones, glucagon, insulin and somatostatin, interactwith specific pancreatic cell types to modulate the secretory response.Although insulin secretion is predominantly controlled by blood glucoselevels, somatostatin inhibits glucose-mediated insulin secretion.

[0007] The human hormone glucagon is a polypeptide hormone produced inpancreatic A-cells. The hormone belongs to a multi-gene family ofstructurally related peptides that include secretin, gastric inhibitorypeptide, vasoactive intestinal peptide and glicenfin. These peptidesvariously regulate carbohydrate metabolism, gastrointestinal motilityand secretory processing. However, the principal recognized actions ofpancreatic glucagon are to promote hepatic glycogenolysis andglyconeogenesis, resulting in an elevation of blood sugar levels. Inthis regard, the actions of glucagon are counter regulatory to those ofinsulin and may contribute to the hyperglycemia that accompaniesDiabetes mellitus (Lund et al. (1982) PNAS, 79:345-349).

[0008] Preproglucagon, the zymogen form of glucagon, is translated froma 360 base pair gene and is processed to form proglucagon (Lund, et al.,supra). Patzelt, et al. (Nature, 282:260-266 (1979)) demonstrated thatproglucagon is further processed into glucagon and a second peptide.Later experiments demonstrated that proglucagon is cleaved carboxyl toLys-Arg or Arg-Arg residues (Lund et al., supra; and Bell et al. (1983)Nature 302:716-718). Bell et al. also discovered that proglucagoncontained three discrete and highly homologous peptide regions whichwere designated glucagon, glucagon-like peptide 1 (GLP-1), andglucagon-like peptide 2 (GLP-2). GLP-1 has attracted increasingattention as a humoral stimulus of insulin secretion. In humans, this29-amino acid peptide, cleaved from proglucagon by cells of theintestinal mucosa, is released into the circulation after nutrientintake (Holst et al. (1987) FEBS Lett 211:169; Orskov et al. (1987)Diabetologia 30:874; Conlon J (1988) Diabetologia 31:563).

[0009] GLP-1 has been found to be a glucose-dependent insulinotropicagent (Gutniak et al. (1992) N. Engl. J. Bled. 326:1316-1322). GLP-1 isnow known to stimulate insulin secretion (insulinotropic action) causingglucose uptake by cells which decreases serum glucose levels (see, e.g.,Mojsov, S., Int. J. Peptide Protein Research, 40:333-343 (1992)). Forinstance, it has been shown to be a potent insulin secretagogue inexperimental models and when infused into humans (Gutniak et al., supra;Mojsov et al. (1988) J Clin Invest 79:616; Schmidt et al. (1985)Diabetologia 28:704; and Kreymann et al. (1987) Lancet 2:1300). Thus,GLP-1 is a candidate for the role of an “incretin”, having augmentaryeffects on glucose-mediated insulin release.

[0010] It is also noted that numerous GLP-1 analogs have beendemonstrated which demonstrate insulinotropic action are known in theart. These variants and analogs include, for example, GLP-1(7-36),Gln₉-GLP-1(7-37), D-Gln₉-GLP-1(7-37), acetyl-Lys₉-GLP-1(7-37),Thr₁₆-Lys₁₈-GLP-1(7-37), and Lys₁₈-GLP-1(7-37). Derivatives of GLP-1including, for example, acid addition salts, carboxylate salts, loweralkyl esters, and amides (see, e.g., WO91/11457).

OBJECTS OF THE INVENTION

[0011] It is one object of this invention to provide improved methodsfor reducing in animal subjects (including humans) in need of suchtreatment at least one of insulin resistance, hyperinsulinemia, andhyperglycemia and abating Type II diabetes. Another object is to provideimproved methods for reducing at least one of body fat stores,hyperlipidemia, hyperlipoproteinemia, and for abating atherosclerosis.It is another object of this invention to provide methods forinterfering with glucose and/or lipid metabolism in a manner beneficialto the host.

[0012] It is yet another object of this invention to provide improvedmethods for the long-term reduction and abatement of at least one of theforegoing disorders based on a therapeutic regimen administered over theshort-term.

[0013] It is still another object of the present invention to-provide amethod for regulating, and altering on a long term basis, the glucoseand lipogenic responses of vertebrate animals, including humans.

[0014] In particular, it is an object of the invention to providemethods for producing long lasting beneficial changes in one or more ofthe following: the sensitivity of the cellular response of a species toinsulin (reduction of insulin resistance), blood insulin levels,hyperinsulinemia, blood glucose levels, the amount of body fat stores,blood lipoprotein levels, and thus to provide effective treatments fordiabetes, obesity and/or atherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a diagrammatic representation of the synthesis of a boroproline compound.

[0016]FIG. 2 is a glucose tolerance curve which shows that a singleinjection of PBP-1 improves glucose levels in blood. The glucoseconcentration is measured before and at 30-minute intervals after thetest dose of glucose. This figure demonstrates that a single injectionof PBP-1 potentiates the response to a sub-therapeutic dose of GLP-1.

[0017]FIG. 3 shows that a single injection of PBP-2 improves glucoselevels in blood.

[0018]FIG. 4 shows that treatment with PBP-3 under “chronic” conditionsalso results in lowering of the blood sugar levels.

[0019]FIGS. 5A and 5B compare the ability of Pro-boro-pro to lowerplasma glucose levels in GLP-1 receptor −/− transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Glucose-induced insulin secretion is modulated by a number ofhormones and neurotransmitters. In particular, two gut hormones,glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP) areinsulinotropic agents, e.g., being agents which can stimulate, or causethe stimulation of, the synthesis or expression of the hormone insulin,are thus called gluco-incretins (Dupre, in The Endocrine Pancreas, E.Samois Ed. (Raven Press, New York, (1991), 253-281); and Ebert et al.(1987) Diabetes Metab. Rev. p3). Glucagon-like peptide-1 is aglucoincretin both in man and other mammals (Dupre et al. supra, andKreymann et al. (1987) Lancet 2:300). It is part of the preproglucagonmolecule (Bell et al. (1983) Nature 304:368) which is proteolyticallyprocessed in intestinal L cells to GLP-1(1-37) and GLP-1(7-36)amide orGLP-1(7-37) (Mojsov et al. (1986) J. Biol. Chem. 261:11880; and Habeneret al.: The Endocrine Pancreas, E. Samois Ed. (Raven Press, New York(1991), 53-71). Only the truncated forms of GLP-1 are biologicallyactive and both have identical effects on insulin secretion in betacells (Mojsov et al. (1987) J. Clin. Invest 79:616; and Weir et al.(1989) Diabetes 38:338). They are the most potent gluco-incretins so fardescribed and are active at concentrations as low as one to tenpicomolar.

[0021] The metabolic fate of exogenous GLP-1 has been studied innondiabetic and type II diabetic subjects. Subcutaneous and intravenousGLP-1 are both rapidly degraded in a time-dependent manner, forinstance, having a half-life in diabetic patients of substantially lessthan 30 minutes. See, for example, Deacon et al. (1995) Diabetes44:1126-1131.

i. Overview of the Invention

[0022] The present invention provides methods and compositions formodification and regulation of glucose and lipid metabolism, generallyto reduce insulin resistance, hyperglycemia, hyperinsulinemia, obesity,hyperlipidemia, hyperlipoprotein-emia (such as chylomicrons, VLDL andLDL), and to regulate body fat and more generally lipid stores, and,more generally, for the improvement of metabolism disorders, especiallythose associated with diabetes, obesity and/or atherosclerosis. Asdescribed in greater detail below, the subject method includes theadministration, to an animal, of a composition including one or moredipeptidylpeptidase inhibitors, especially inhibitors of thedipeptidylpeptidase IV (DPIV) enzyme or other enzyme of similarspecificity, which are able to inhibit the proteolysis of GLP-1 andaccordingly increase the plasma half-life of that hormone.

[0023] Preferably, the compounds utilized in the subject method willproduce an EC50 for the desired biological effect of at least one, two,three and even four orders of magnitude less than the EC50 for thatcompound as an immunosuppressant. Indeed, a salient feature of suchcompounds as the peptidyl boronates is that the inhibitors can produce,for example, an EC50 for inhibition of glucose tolerance in thenanomolar or less range, whereas the compounds have EC50's forimmunosuppression in the μM or greater range. Thus, a favorabletherapeutic index can be realized with respect to the unwantedsideeffect of immunosuppression.

[0024] While not wishing to bound by any particular theory, it isobserved that compounds which inhibit DPIV are, correlatively, able toimprove glucose tolerance, though not necessarily through mechanismsinvolving DPIV inhibition per se. Indeed, the results described inExample 6 (and FIG. 5) demonstrating an effect in mice lacking a GLP-1receptor suggest that the subject method may not include a mechanism ofaction directly implicating GLP-1 itself, though it has not been ruledout that GLP-1 may have other receptors. However, in light of thecorrelation with DPIV inhibition, in preferred embodiments, the subjectmethod utilizes an agent with a Ki for DPIV inhibition of 1.0 nm orless, more preferably of 0.1 nm or less, and even more preferably of0.01 nM or less. Indeed, inhibitors with Ki values in the picomolar andeven femtamolar range are contemplated. Thus, while the active agentsare described herein, for convience, as “DPIV inhibitors”, it will beunderstood that such nomenclature is not intending to limit the subjectinvention to a particular mechanisim of action.

[0025] For instance, in certian embodiments the method involvesadministration of a DPIV inhibitor, preferably at a predeterminedtime(s) during a 24-hour period, in an amount effective to improve oneor more aberrant indices associated with glucose metabolism disorders(e.g., glucose intolerance, insulin resistance, hyperglycemia,hyperinsulinemia and Type II diabetes).

[0026] In other embodiments, the method involves administration of aDPIV inhibitor in an amount effective to improve aberrant indicesassociated with obesity. Fat cells release the hormone leptin, whichtravels in the bloodstream to the brain and, through leptin receptorsthere, stimulates production of GLP-1. GLP-1, in turn, produces thesensation of being full. The leading theory is that the fat cells ofmost obese people probably produce enough leptin, but leptin may not beable to properly engage the leptin receptors in the brain, and so doesnot stimulate production of GLP-1. There is accordingly a great deal ofresearch towards utilizing preparations of GLP-1 as an apepititesuppressant. The subject method provides a means for increasing thehalf-life of both endogenous and ectopically added GLP-1 in thetreatment of disorders associated with obesity.

[0027] In a more general sense, the present invention provides methodsand compositions for altering the pharmokinetics of a variety ofdifferent polypeptide hormones by inhibiting the proteolysis of one ormore peptide hormones by DPIV or some other proteolytic activity.Post-secretory metabolism is an important element in the overallhomeostasis of regulatory peptides, and the other enzymes involved inthese processes may be suitable targets for pharmacological interventionby the subject method.

[0028] For example, the subject method can be used to increase thehalf-life of other proglucagon-derived peptides, such as glicentin(corresponding to PG 1-69), oxyntomodulin (PG 33-69), glicentin-relatedpancreatic polypeptide (GRPP, PG 1-30), intervening peptide-2 (IP-2, PG111-122amide), and glucagon-like peptide-2 (GLP-2, PG 126-158).

[0029] GLP-2, for example, has been identified as a factor responsiblefor inducing proliferation of intestinal epithelium. See, for example,Drucker et al. (1996) PNAS 93:7911. The subject method can be used aspart of a regimen for treating injury, inflammation or resection ofintestinal tissue, e.g., where enhanced growth and repair of theintestinal mucosal epithelial is desired.

[0030] DPIV has also been implicated in the metabolism and inactivationof growth hormone-releasing factor (GHRF). GHRF is a member of thefamily of homologous peptides that includes glilcagon, secretin,vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI),pituitary adenylate cyclase activating peptide (PACAP), gastricinhibitory peptide (GIP) and helodermin. Kubiak et al. (1994) PeptideRes 7:153. GHRF is secreted by the hypothalamus, and stimulates therelease of growth hormone (GH) from the anterior pituitary. Thus, thesubject method can be used to improve clinical therapy for certaingrowth hormone deficient children, and in clinical therapy of adults toimprove nutrition and to alter body composition (muscle vs. fat). Thesubject method can also be used in veterinary practice, for example, todevelop higher yield milk production and higher yield, leaner livestock.

[0031] Likewise, the DPIV inhibitors of the subject invention can beused to alter the plasma half-life of secretin, VIP, PHI, PACAP, GIPand/or helodermin. Additionally, the subject method can be used to alterthe pharmacokinetics of Peptide YY and neuropeptide Y, both members ofthe pancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

[0032] Another aspect of the present invention relates to pharmaceuticalcompositions of dipeptidylpeptidase inhibitors, particularly DPIVinhibitors, and their uses in treating and/or preventing disorders whichcan be improved by altering the homeostasis of peptide hormones. In apreferred embodiment, the inhibitors have hypoglycemic and antidiabeticactivities, and can be used in the treatment of disorders marked byabberrant glucose metabolism (including storage). In particularembodiments, the compositions of the subject methods are useful asinsulinotropic agents, or to potentiate the insulinotropic effects ofsuch molecules as GLP-1. In this regard, the present method can beuseful for the treatment and/or prophylaxis of a variety of disorders,including one or more of: hyperlipemia, hyperglycemia, obesity, glucosetolerance insufficiency, insulin resistance and diabetic complications.

[0033] In general, the inhibitors of the subject method will be smallmolecules, e.g., with molecular weights less than 7500 amu, preferablyless than 5000 amu, and even more preferably less than 2000 amu and even1000 amu. In preferred embodiments, the inhibitors will be orallyactive.

[0034] In certain embodiments, the subject inhibitors are peptidylcompounds (including peptidomimetics) which are optimized, e.g.,generally by selection of the Cα substituents, for the substratespecificity of the targeted proteolytic activity. These peptidylcompounds will include a functional group, such as in place of thescissile peptide bond, which facilitates inhibition of a serine-,cysteine- or aspartate-type protease, as appropriate. For example, theinhibitor can be a peptidyl α-diketone or a peptidyl α-keto ester, apeptide haloalkylketone, a peptide sulfonyl fluoride, a peptidylboronate, a peptide epoxide, a peptidyl diazomethanes, a peptidylphosphonate, isocoumarins, benzoxazin-4-ones, carbamates, isocyantes,isatoic anhydrides or the like. Such functional groups have bee providedin other protease inhibitors, and general routes for their synthesis areknown. See, for example, Angelastro et al., J. Med Chem. 33:11-13(1990); Bey et al., EPO 363,284; Bey et al., EPO 364,344; Grubb et al.,WO 88/10266; Higuchi et al., EPO 393,457; Ewoldt et al., MolecularImmunology 29(6):713-721 (1992); Hernandez et al., Journal of MedicinalChemistry 35(6): 1121-1129 (1992); Vlasak et al., J. Virology63(5):2056-2062 (1989); Hudig et al., J Immunol 147(4):1360-1368 (1991);Odakc et al., Biochemistry 30(8):2217-2227 (1991); Vijayalakshmi et al.,Biochemistry 30(8):2175-2183 (1991); Kam et al., Thrombosis andHaemostasis 64(1):133-137 (1990); Powers et al., J Cell Biochem39(1):33-46 (1989); Powers et al., Proteinase Inhibitors, Barrett etal., Eds., Elsevier, pp. 55-152 (1986); Powers et al., Biochemistry29(12):3108-3118 (1990); Oweida et al., Thrombosis Research58(2):391-397 (1990); Hudig et al., Molecular Immunology 26(8):793-798(1989); Orlowski et al., Archives of Biochemistry and Biophysics269(1):125-136 (1989); Zunino et al., Biochimica et Biophysica Acta.967(3):331-340 (1988); Kam et al., Biochemistry 27(7):2547-2557 (1988);Parkes et al., Biochem J. 230:509-516 (1985); Green et al., J. Biol.Chem. 256:1923-1928 (1981); Angliker et al., Biochem. J. 241:871-875(1987); Puri et al., Arch. Biochem. Biophys. 27:346-358 (1989); Hanadaet al., Proteinase Inhibitors: Medical and Biological Aspects, Katunumaet al., Eds., Springer-Verlag pp. 25-36 (1983); Kajiwara et al.,Biochem. Int. 15:935-944 (1987); Rao et al., Thromb. Res. 47:635-637(1987); Tsujinaka et al., Biochem. Biophys. Res. Commun. 153:1201-1208(1988)). See also U.S. Patents Bachovchin et al. 4,935,493; Bachovchinet al. U.S. Pat. No. 5,462,928; Powers et al. U.S. Pat. No. 5,543,396;Hanko et al. U.S. Pat. No. 5,296,604; and the PCT publication of FerringPCT/GB94/02615.

[0035] In other embodiments, the inhibitor is a non-peptidyl compound,e.g., which can be identified by such drug screening assays as describedherein. These inhibitors can be, merely to illustrate, syntheticorganis, natural products, nucleic acids or carbohydrates.

[0036] A representative class of compounds for use in the method of thepresent invention are represented by the general formula;

[0037] wherein

[0038] A represents a 4-8 membered heterocycle including the N and theCα carbon;

[0039] Z represents C or N;

[0040] W represents a functional group which reacts with an active siteresidue of the targeted protease, as for example, —CN, —CH═NR₅,

[0041] R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

[0042] R₂ is absent or represents one or more substitutions to the ringA, each of which can independently be a halogen, a lower alkyl, a loweralkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, aformate, or a ketone), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an amino, an acylamino, an amido, acyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)S—(CH₂)_(m)—R₇;

[0043] if X is N, R₃ represents hydrogen, if X is C, R₃ representshydrogen or a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl,a carbonyl (such as a carboxyl, an ester, a formate, or a ketone), athiocarbonyl (such as a thioester, a thioacetate, or a thioformnate), anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇;

[0044] R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m—R₇, —(CH₂)n—OH, —(CH₂)n—O-alkyl, —(CH₂)n O-alkenyl,—(CH₂)n—O-alkynyl, —(CH₂)n—O—(CH₂)m—R₇, —(CH₂)n—SH, —(CH₂)n—S-alkyl,—(CH₂)n—S-alkenyl, —(CH₂)n—S-alkylnyl, —(CH₂)n—S—(CH₂)_(m)—R₇,—C(O)C(O)NH₂, —C(O)C(O)OR′₇;

[0045] R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl,an aryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

[0046] R₇ represents, for each occurrence, a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

[0047] R′₇ represents, for each occurrence, hydrogen, or a substitutedor unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and

[0048] Y₁ and Y₂ can independently or together be OH, or a group capableof being hydrolyzed to a hydroxyl group, including cyclic derivativeswhere Y₁ and Y₂ are connected via a ring having from 5 to 8 atoms in thering structure (such as pinacol or the like),

[0049] R₅₀ represents O or S;

[0050] R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇;

[0051] R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

[0052] X₁ represents a halogen;

[0053] X₂ and X₃ each represent a hydrogen or a halogen

[0054] m is zero or an integer in the range of 1 to 8; and n is aninteger in the range of 1 to 8.

[0055] In preferred embodiments, the ring A is a 5, 6 or 7 memberedring, e.g., represented by the formula

[0056] and more preferably a 5 or 6 membered ring. The ring may,optionally, be further substituted.

[0057] In preferred embodiments, W represents

[0058] In preferred embodiments, R1 is

[0059] wherein R36 is a small hydrophobic group, e.g., a lower alkyl ora halogen and R38 is hydrogen, or, R36 and R37 together form a 4-7membered heterocycle including the N and the Cα carbon, as defined for Aabove; and R40 represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group

[0060] In preferred embodiments, R2 is absent, or represents a smallhydrophobic group such as a lower alkyl or a halogen.

[0061] In preferred embodiments, R3 is a hydrogen, or a smallhydrophobic group such as a lower alkyl or a halogen.

[0062] In preferred embodiments, R5 is a hydrogen, or a halogentatedlower alkyl.

[0063] In preferred embodiments, X1 is a fluorine, and X2 and X3, ifhalogens, are fluorine.

[0064] Also deemed as equivalents are any compounds which can behydrolytically converted into any of the aforementioned compoundsincluding boronic acid esters and halides, and carbonyl equivalentsincluding acetals, hemiacetals, ketals, and hemiketals, and cyclicdipeptide analogs.

[0065] Longer peptide sequences are needed for the inhibition of certainproteases and improve the specificity of the inhibition in some cases.

[0066] In preferred embodiments, the subject method utilizes, as a DPIVinhibitor, a boronic acid analogs of an amino acid. For example, thepresent invention contemplates the use of boro-prolyl derivatives in thesubject method. Exemplary boronic acid derived inhibitors of the presentinvention are represented by the general formula:

[0067] wherein

[0068] R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a terminally linked peptide or peptide analog, or

[0069] R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl,an aryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

[0070] R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or aheterocycle;

[0071] R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

[0072] or R₈ and R₉ taken together with the N atom to which they areattached complete a heterocyclic ring having from 4 to 8 atoms in thering structure;

[0073] R₁₁ and R₁₂ each independently represent hydrogen, a alkyl, or apharmaceutically acceptable salt, or R₁₁ and R₁₂ taken together with theO—B—O atoms to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

[0074] m is zero or an integer in the range of 1 to 8; and n is aninteger in the range of 1 to 8.

[0075] In other embodiments, the subject DPIV inhibitors include analdehyde analogs of proline or prolyl derivatives. Exemplaryaldehyde-derived inhibitors of the present invention are represented bythe general formula:

[0076] wherein

[0077] R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a terminally linked peptide or peptide analog, or

[0078] C—

[0079] R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl,an aryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

[0080] R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or aheterocycle;

[0081] R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

[0082] or R₈ and R₉ taken together with the N atom to which they areattached complete a heterocyclic ring having from 4 to 8 atoms in thering structure; and

[0083] m is zero or an integer in the range of 1 to 8; and n is aninteger in the range of 1 to 8.

[0084] In yet further embodiments, the subject DPIV inhibitors arehalo-methyl ketone analogs of an amino acid. Exemplary inhibitors ofthis class include compounds represented by the general formula:

[0085] wherein

[0086] R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a terminally linked peptide or peptide analog, or

[0087] C—

[0088] R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl,an aryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

[0089] R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or aheterocycle;

[0090] R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇,

[0091] or R₈ and R₉ taken together with the N atom to which they areattached complete a heterocyclic ring having from 4 to 8 atoms in thering structure;

[0092] X₁, X₂ and X₃ each represent a hydrogen or a halogen; and

[0093] m is zero or an integer in the range of 1 to 8; and n is aninteger in the range of 1 to 8.

[0094] In preferred embodiments, the DPIV inhibitor is a peptide orpeptidomimetic including a prolyl group or analog thereof in the P1specificity position, and a nonpolar amino acid in the P2 specificityposition, e.g., a nonpolar amino acid such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan or methionine, oran analog thereof. For example, the DPIV inhibitor may include anAla-Pro or Pro-Pro dipeptide sequence or equivalent thereof, and berepresented in the general formulas:

[0095] In preferred embodiments, the ring A is a 5, 6 or 7 memberedring, e.g., represented by the formula

[0096] In preferred embodiments, R32 is a small hydrophobic group, e.g.,a lower alkyl or a halogen.

[0097] In preferred embodiments, R30 represents a C-terminally linkedamino acid residue or amino acid analog, or a C-terminally linkedpeptide or peptide analog, or an amino-protecting group.

[0098] In preferred embodiments, R2 is absent, or represents a smallhydrophobic group such as a lower alkyl or a halogen.

[0099] In preferred embodiments, R3 is a hydrogen, or a smallhydrophobic group such as a lower alkyl or a halogen.

[0100] Another representative class of compounds for use in the subjectmethod include peptide and peptidomimetics of (D)-Ala-(L)-Ala, e.g.,preserving the diasteromeric orientation. Such inhibitors includecompounds represented by the general formula:

[0101] wherein

[0102] W represents a functional group which reacts with an active siteresidue of the targeted protease, as for example, —CN, —CH═NR₅,

[0103] R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

[0104] R₃ represents hydrogen or a halogen, a lower alkyl, a loweralkenyl, a lower alkynyl, a carbonyl (such as a carboxyl, an ester, aformate, or a ketone), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an amino, an acylamino, an amido, acyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)-R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇;

[0105] R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m—R₇, —(CH₂)n—OH, —(CH₂)n—O-alkyl, —(CH₂)n—O-alkenyl,—(CH₂)n—O-alkynyl, —(CH₂)n—O—(CH₂)m—R₇, —(CH₂)n—SH, —(CH₂)n—S-alkyl,—(CH₂)n—S-alkenyl, —(CH₂)n—S-alkynyl, —(CH₂)n—S—(CH₂)m—R₇, —C(O)C(O)NH₂,—C(O)C(O)OR′₇;

[0106] R6 represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl,an aryl, —(CH2)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—s—(CH₂)_(m)—R₇,

[0107] R₇ represents, for each occurrence, a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

[0108] R′₇ represents, for each occurrence, hydrogen, or a substitutedor unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle;

[0109] R₆₁ and R₆₂, indepedently, represent small hydrophobic groups;

[0110] Y₁ and Y₂ can independently or together be OH, or a group capableof being hydrolyzed to a hydroxyl group, including cyclic derivativeswhere Y₁ and Y₂ are connected via a ring having from 5 to 8 atoms in thering structure (such as pinacol or the like),

[0111] R₅₀ represents O or S;

[0112] R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇;

[0113] R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

[0114] X₁ represents a halogen;

[0115] X₂ and X₃ each represent a hydrogen or a halogen

[0116] m is zero or an integer in the range of 1 to 8; and n is aninteger in the range of 1 to 8.

[0117] In preferred embodiments, R₁ is

[0118] wherein R36 is a small hydrophobic group, e.g., a lower alkyl ora halogen and R38 is hydrogen, or, R36 and R37 together form a 4-7membered heterocycle including the N and the Cα carbon, as defined for Aabove; and R40 represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group

[0119] In preferred embodiments, R3 is a hydrogen, or a smallhydrophobic group such as a lower alkyl or a halogen.

[0120] In preferred embodiments, R5 is a hydrogen, or a halogentatedlower alkyl.

[0121] In preferred embodiments, X1 is a fluorine, and X2 and X3, ifhalogens, are fluorine.

[0122] In preferred embodiments, R₆₁ and R62, independently, representlow alkyls, such as methyl, ethyl, propyl, isopropyl, tert-butyl or thelik.;

[0123] Also included are such peptidomimetics as olefins, phosphonates,aza-amino acid analogs and the like.

[0124] Also deemed as equivalents are any compounds which can behydrolytically converted into any of the aforementioned compoundsincluding boronic acid esters and halides, and carbonyl equivalentsincluding acetals, hemiacetals, ketals, and hemiketals, and cyclicdipeptide analogs.

[0125] As used herein, the definition of each expression, e.g. alkyl, m,n, etc., when it occurs more than once in any structure, is intended tobe independent of its definition elsewhere in the same structure.

[0126] The pharmaceutically acceptable salts of the subject compoundsinclude the conventional nontoxic salts or quaternary ammonium salts ofthe compounds, e.g., from non-toxic organic or inorganic acids. Forexample, such conventional nontoxic salts include those derived frominorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

[0127] The pharmaceutically acceptable salts of the present inventioncan be synthesized from the subject compound which contain a basic oracid moiety by conventional chemical methods. Generally, the salts areprepared by reacting the free base or acid with stoichiometric amountsor with an excess of the desired salt-forming inorganic or organic acidor base in a suitable solvent. The pharmaceutically acceptable salts ofthe acids of the subject compounds are also readily prepared byconventional procedures such as treating an acid of Formula I with anappropriate amount of a base such as an alkali or alkaline earth methylhydroxide (e.g. sodium, potassium, lithium, calcium or magnesium) or anorganic base such as an amine, piperidine, pyrrolidine, benzylamine andthe like, or a quaternary ammonium hydroxide such as tetramethylammoniumhydroxide and the like.

[0128] Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g. the ability to inhibit proteolysis ofGLP-1 or other peptide hormone or precursor thereof), wherein one ormore simple variations of substituents are made which do not adverselyaffect the efficacy of the compound in use in the contemplated method.In general, the compounds of the present invention may be prepared bythe methods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants which arein themselves known, but are not mentioned here.

ii. Definitions

[0129] For convenience, before further description of the presentinvention, certain terms employed in the specification, examples, andappended claims are collected here.

[0130] The term “alkyl” refers to the radical of saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In preferredembodiments, a straight chain or branched chain alkyl has 30 or fewercarbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀for branched chain), and more preferably 20 or fewer. Likewise,preferred cycloalkyls have from 3-10 carbon atoms in their ringstructure, and more preferably have 5, 6 or 7 carbons in the ringstructure.

[0131] Moreover, the term “alkyl” (or “lower alkyl”) as used throughoutthe specification and claims is intended to include both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an ester, aformyl, or a ketone), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate,a phosphinate, an amino, an amido, an amidine, an imine, a cyano, anitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate. For instance, thesubstituents of a substituted alkyl may include substituted andunsubstituted forms of amino, azido, imino, amido, phosphoryl (includingphosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido,sulfamoyl and sulfonate), and silyl groups, as well as ethers,alkylthios, carbonyls (including ketones, aldehydes, carboxylates, andesters), —CF₃, —CN and the like. Exemplary substituted alkyls aredescribed below. Cycloalkyls can be further substituted with alkyls,alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls,—CF₃, —CN, and the like.

[0132] The term “aralkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

[0133] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous in length and possible substitution to the alkylsdescribed above, but that contain at least one double or triple bondrespectively.

[0134] Unless the number of carbons is otherwise specified, “loweralkyl” as used herein means an alkyl group, as defined above, but havingfrom one to ten carbons, more preferably from one to six carbon atoms inits backbone structure. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

[0135] The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromaticmoieties, —CF₃, —CN, or the like. The term “aryl” also includespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings (the rings are “fusedrings”) wherein at least one of the rings is aromatic, e.g., the othercyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls.

[0136] The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobehzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

[0137] The terms “polycyclyl” or “polycyclic group” refer to two or morerings (e.g., cycloalkyls, cycloalkenyls cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

[0138] The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

[0139] The term “heteroatom” as used herein means an atom of any elementother than carbon or hydrogen. Preferred heteroatoms are nitrogen,oxygen, sulfur and phosphorous.

[0140] As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

[0141] The terms “amine” and “amino” are art recognized and refer toboth unsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

[0142] wherein R₉, R₁₀ and R′₁₀ each independently represent a hydrogen,an alkyl, an alkenyl, —(CH₂)_(m)—R₈, or R₉ and R₁₀ taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R₈ represents an aryl, a cycloalkyl,a cycloalkenyl, a heterocycle or a polycycle; and m is zero or aninteger in the range of 1 to 8. In preferred embodiments, only one of R₉or R₁₀ can be a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do notform an imide. In even more preferred embodiments, R₉ and R₁₀ (andoptionally R′₁₀) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R₈. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₉ and R₁₀is an alkyl group.

[0143] The term “acylamino” is art-recognized and refers to a moietythat can be represented by the general formula:

[0144] wherein R9 is as defined above, and R′₁₁ represents a hydrogen,an alkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as definedabove.

[0145] The term “amido” is art recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

[0146] wherein R₉, R₁₀ are as defined above. Preferred embodiments ofthe amide will not include imides which may be unstable.

[0147] The term “alkylthio” refers to an alkyl group, as defined above,having a sulfur radical. attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

[0148] The term “carbonyl” is art recognized and includes such moietiesas can be represented by the general formula:

[0149] wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′₁₁ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

[0150] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

[0151] The term “sulfonate” is art recognized and includes a moiety thatcan be represented by the general formula:

[0152] in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, oraryl.

[0153] The term “sulfate” is art recognized and includes a moiety thatcan be represented by the general formula:

[0154] in which R₄₁ is as defined above.

[0155] The term “sulfonamido” is art recognized and includes a moietythat can be represented by the general formula:

[0156] in which R₉ and R′₁₁ are as defined above.

[0157] The term “sulfamoyl” is art-recognized and includes a moiety thatcan be represented by the general formula:

[0158] in which R₉ and R₁₀ are as defined above.

[0159] The terms “sulfoxido” or “sulfinyl”, as used herein, refers to amoiety that can be represented by the general formula:

[0160] in which R₄₄ is selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

[0161] A “phosphoryl” can in general be represented by the formula:

[0162] wherein Q₁ represented S or O, and R₄₆ represents hydrogen, alower alkyl or an aryl. When used to substitute, e.g., an alkyl, thephosphoryl group of the phosphorylalkyl can be represented by thegeneral formula:

[0163] wherein Q₁ represented S or O, and each R₄₆ independentlyrepresents hydrogen, a lower alkyl or an aryl, Q₂ represents O, S or N.When Q₁ is an S, the phosphoryl moiety is a “phosphorothioate”.

[0164] A “phosphoramidite” can be represented in the general formula:

[0165] wherein R₉ and R₁₀ are as defined above, and Q₂ represents O, Sor N.

[0166] A “phosphonamidite” can be represented in the general formula:

[0167] wherein R₉ and R₁₀ are as defined above, Q₂ represents O, S or N,and R₄₈ represents a lower alkyl or an aryl, Q₂ represents O, S or N.

[0168] A “selenoalkyl” refers to an alkyl group having a substitutedseleno group attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

[0169] Analogous substitutions can be made to alkenyl and alkynyl groupsto produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

[0170] It will be understood that “substitution” or “substituted with”includes the implicit proviso that such substitution is in accordancewith permitted valence of the substituted atom and the substituent, andthat the substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

[0171] As used herein, the term “substituted” is contemplated to includeall permissible substituents of organic compounds. In a broad aspect,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described hereinabove. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalencies of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

[0172] A “small” substituent is one of 10 atoms or less.

[0173] By the terms “amino acid residue” and “peptide residue” is meantan amino acid or peptide molecule without the —OH of its carboxyl group.In general the abbreviations used herein for designating the amino acidsand the protective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature (see Biochemistry (1972)11:1726-1732). For instance Met, Ile, Leu, Ala and Gly represent“residues” of methionine, isoleucine, leucine, alanine and glycine,respectively. By the residue is meant a radical derived from thecorresponding α-amino acid by eliminating the OH portion of the carboxylgroup and the H portion of the α-amino group. The term “amino acid sidechain” is that part of an amino acid exclusive of the —CH(NH₂)COOHportion, as defined by K. D. Kopple, “Peptides and Amino Acids”, W. A.Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33; examples ofsuch side chains of the common amino acids are —CH₂CH₂SCH₃ (the sidechain of methionine), —CH₂(CH₃)—CH₂CH₃ (the side chain of isoleucine),—CH₂CH(CH₃)₂ (the side chain of leucine) or H— (the side chain ofglycine).

[0174] For the most part, the amino acids used in the application ofthis invention are those naturally occurring amino acids found inproteins, or the naturally occurring anabolic or catabolic products ofsuch amino acids which contain amino and carboxyl groups. Particularlysuitable amino acid side chains include side chains selected from thoseof the following amino acids: glycine, alanine, valine, cysteine,leucine, isoleucine, serine, threonine, methionine, glutamic acid,aspartic acid, glutamine, asparagine, lysine, arginine, proline,histidine, phenylalanine, tyrosine, and tryptophan, and those aminoacids and amino acid analogs which have been identified as constituentsof peptidylglycan bacterial cell walls.

[0175] The term amino acid residue further includes analogs, derivativesand congeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g. modifiedwith an N-terminal or C-terminal protecting group). For example, thepresent invention contemplates the use of amino acid analogs wherein aside chain is lengthened or shortened while still providing a carboxyl,amino or other reactive precursor functional group for cyclization, aswell as amino acid analogs having variant side chains with appropriatefunctional groups). For instance, the subject compound can include anamino acid analog such as, for example, cyanoalanine, canavanine,djenkolic acid, norleucine, 3-phosphoserine, homoserine,dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine,3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyricacid. Other naturally occurring amino acid metabolites or precursorshaving side chains which are suitable herein will be recognized by thoseskilled in the art and are included in the scope of the presentinvention.

[0176] Also included are the (D) and (L) stereoisomers of such aminoacids when the structure of the amino acid admits of stereoisomericforms. The configuration of the amino acids and amino acid residuesherein are designated by the appropriate symbols (D), (L) or (DL),furthermore when the configuration is not designated the amino acid orresidue can have the configuration (D), (L) or (DL). It will be notedthat the structure of some of the compounds of this invention includesasymmetric carbon atoms. It is to be understood accordingly that theisomers arising from such asymmetry are included within the scope ofthis invention. Such isomers can be obtained in substantially pure formby classical separation techniques and by sterically controlledsynthesis. For the purposes of this application, unless expressly notedto the contrary, a named amino acid shall be construed to include boththe (D) or (L) stereoisomers.

[0177] The phrase “protecting group” as used herein means substituentswhich protect the reactive functional group from undesirable chemicalreactions. Examples of such protecting groups include esters ofcarboxylic acids and boronic acids, ethers of alcohols and acetals andketals of aldehydes and ketones. For instance, the phrase “N-terminalprotecting group” or “amino-protecting group” as used herein refers tovarious amino-protecting groups which can be employed to protect theN-terminus of an amino acid or peptide against undesirable reactionsduring synthetic procedures. Examples of suitable groups include acylprotecting groups such as, to illustrate, formyl, dansyl, acetyl,benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromaticurethane protecting groups as, for example, benzyloxycarbonyl (Cbz); andaliphatic urethane protecting groups such as t-butoxycarbonyl (Boc) or9-Fluorenylmethoxycarbonyl (FMOC).

[0178] As noted above, certain compounds of the present invention mayexist in particular geometric or stereoisomeric forms. The presentinvention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

[0179] If, for instance, a particular enantiomer of a compound of thepresent. invention is desired, it may be prepared by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts are formed withan appropriate optically-active acid or base, followed by resolution ofthe diastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

[0180] For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 67th Ed., 1986-87, insidecover. Also for purposes of this invention, the term “hydrocarbon” iscontemplated to include all permissible compounds having at least onehydrogen and one carbon atom. In a broad aspect, the permissiblehydrocarbons include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic organic compoundswhich can be substituted or unsubstituted.

[0181] A compound is said to have an “insulinotropic activity” if it isable to stimulate, or cause the stimulation of, the synthesis orexpression of the hormone insulin.

iii. Exemplary Formulations

[0182] A. Agonism of GLP-1 Effects

[0183] The inhibitors useful in the subject methods possess, in certainembodiments, the ability to lower blood glucose levels, to relieveobesity, to alleviate impaired glucose tolerance, to inhibit hepaticglucose neogenesis, and to lower blood lipid levels and to inhibitaldose reductase. They are thus useful for the prevention and/or therapyof hyperglycemia, obesity, hyperlipidemia, diabetic complications(including retinopathy, nephropathy, neuropathy, cataracts, coronaryartery disease and arteriosclerosis) and furthermore for obesity-relatedhypertension and osteoporosis.

[0184] Diabetes mellitus is a disease characterized by hyperglycemiaoccurring from a relative or absolute decrease in insulin secretion,decreased insulin sensitivity or insulin resistance. The morbidity andmortality of this disease result from vascular, renal, and neurologicalcomplications. An oral glucose tolerance test is a clinical test used todiagnose diabetes. In an oral glucose tolerance test, a patient'sphysiological response to a glucose load or challenge is evaluated.After ingesting the glucose, the patient's physiological response to theglucose challenge is evaluated. Generally, this is accomplished bydetermining the patient's blood glucose levels (the concentration ofglucose in the patient's plasma, serum or whole blood) for severalpredetermined points in time.

[0185] As described in the appended examples, we demonstrate that, invivo, high affinity inhibitors of DPIV are biologically active withrespect to regulation of glucose metabolism. For example, a singleinjection of the inhibitor Pro-boro-Pro (see examples for structure) wasalone sufficient to improve glucose control. A single injection ofPro-boro-Pro was also observed to potentiate the response to asubtherapeutic dose of GLP-1. We have also observed that chronic (>5days) treatment with Pro-boro-Pro alone lowers both fasting bloodsugars, and the glycemic excursion to oral glucose challenge.

[0186] As indicated above, the inhibitors useful in the subject methodcan be peptide- or peptidomimetic-derived inhibitors of the targetproteolytic activity, or can be a non-peptide compound identified, e.g.,by drug screening assays described herein. With respect to DPIVinhibitors, a salient feature of the subject method is the unexpectedfinding that certain DPIV inhibitors have antidiabetic activity atconcentrations significantly lower than the EC50 of the compound as animmunosuppressant. Thus, an animal can be dosed under a regimen designedto provide a blood serum concentration of inhibitor at or about the EC50for antidiabetic effects, and still be sufficiently below the EC50 forimmunosuppressive activity so as to avoid complications resulting fromthat activity. Indeed, for certain of the subject inhibitors, it isanticipated that dosing can be at least an order of magnitude or moregreater than the antidiabetic EC50, yet still remain sufficiently belowa dose producing any significant immunosuppression.

[0187] As discussed further below, a variety of assays are available inthe art for identifying potential inhibitors of DPIV and the like, aswell as assessing the various biological activities (includingside-effects and toxicity) of such an inhibitor.

[0188] B. Agonism of other Peptide Hormones

[0189] In another embodiment, the subject agents can be used to agonize(e.g., mimic or potentiate) the activity of other polypeptide hormones.

[0190] To illustrate, the present invention provides a method foragonizing the action of GLP-2. It has been determined that GLP-2 acts asa trophic agent, to promote growth of gastrointestinal tissue. Theeffect of GLP-2 is marked particularly by increased growth of the smallbowel, and is therefore herein referred to as an “intestinotrophic”effect.

[0191] In still other embidiments, the subject method can be used toincrease the half-life of other proglucagon-derived peptides, such asglicentin, oxyntomodulin, glicentin-related pancreatic polypeptide(GRPP), and/or intervening peptide-2 (IP-2). For example, glicentin hasbeen demonstrated to cause proliferation of intestinal mucosa and alsoinhibits a peristalsis of the stomach, and has thus been elucidated asuseful as a therapeutic agent for digestive tract diseases, thus leadingto the present invention.

[0192] Thus, in one aspect, the present invention relates to therapeuticand related uses of DPIV inhibitors for promoting the growth andproliferation of gastrointestinal tissue, most particularly small boweltissue. For instance, the subject method can be used as part of aregimen for treating injury, inflammation or resection of intestinaltissue, e.g., where enhanced growth and repair of the intestinal mucosalepithelial is desired.

[0193] With respect to small bowel tissue, such growth is measuredconveniently as a increase in small bowel mass and length, relative toan untreated control. The effect of subject inhibitors on small bowelalso manifests as an increase in the height of the crypt plus villusaxis. Such activity is referred to herein as an “intestinotrophic”activity. The efficacy of the subject method may also be detectable asan increase in crypt cell proliferation and/or a decrease in small bowelepithelium apoptosis. These cellular effects may be noted mostsignificantly in relation to the jejunum, including the distal jejunumand particularly the proximal jejunum, and also in the distal ileum. Acompound is considered to have “intestinotrophic effect” if a testanimal exhibits significantly increased small bowel weight, increasedheight of the crypt plus villus axis, or increased crypt cellproliferation or decreased small bowel epithelium apoptosis when treatedwith the compound (or genetically engineered to express it themselves).A model suitable for determining such gastrointestinal growth isdescribed by U.S. Pat. No. 5,834,428.

[0194] In general, patients who would benefit from either increasedsmall intestinal mass and consequent increased small bowel mucosalfunction are candidates for treatment by the subject method. Particularconditions that may be treated include the various forms of sprueincluding celiac sprue which results from a toxic reaction to α-gliadinfrom wheat, and is marked by a tremendous loss of villae of the bowel;tropical sprue which results from infection and is marked by partialflattening of the villae; hypogammaglobulinemic sprue which is observedcommonly in patients with common variable immunodeficiency orhypogammaglobulinemia and is marked by significant decrease in villusheight. The therapeutic efficacy of the treatment may be monitored byenteric biopsy to examine the villus morphology, by biochemicalassessment of nutrient absorption, by patient weight gain, or byamelioration of the symptoms associated with these conditions. Otherconditions that may be treated by the subject method, or for which thesubject method may be useful prophylactically, include radiationenteritis, infectious or post-infectious enteritis, regional enteritis(Crohn's disease), small intestinal damage due to toxic or otherchemotherapeutic agents, and patients with short bowel syndrome.

[0195] More generally, the present invention provides a therapeuticmethod for treating digestive tract diseases. The term “digestive tract”as used herein means a tube through which food passes, including stomachand intestine. The term “digestive tract diseases” as used herein meansdiseases accompanied by a qualitative or quantitative abnormality in thedigestive tract mucosa, which include, e.g., ulceric or inflammatorydisease; congenital or acquired digestion and absorption disorderincluding malabsorption syndrome; disease caused by loss of a mucosalbarrier function of the gut; and protein-losing gastroenteropathy. Theulceric disease includes, e.g., gastric ulcer, duodenal ulcer, smallintestinal ulcer, colonic ulcer and rectal ulcer. The inflammatorydisease include, e.g., esophagitis, gastritis, duodenitis, enteritis,colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiationenteritis, radiation colitis, radiation proctitis, enteritis andmedicamentosa. The malabsorption syndrome includes the essentialmalabsorption syndrome such as disaccharide-decomposing enzymedeficiency, glucose-galactose malabsorption, fractose malabsorption;secondary malabsorption syndrome, e.g., the disorder caused by a mucosalatrophy in the digestive tract through the intravenous or parenteralnutrition or elemental diet, the disease caused by the resection andshunt of the small intestine such as short gut syndrome, cul-de-sacsyndrome; and indigestible malabsorption syndrome such as the diseasecaused by resection of the stomach, e.g., dumping syndrome.

[0196] The term “therapeutic agent for digestive tract diseases” as usedherein means the agents for the prevention and treatment of thedigestive tract diseases, which include, e.g., the therapeutic agent fordigestive tract ulcer, the therapeutic agent for inflammatory digestivetract disease, the therapeutic agent for mucosal atrophy in thedigestive tract and the therapeutic agent for digestive tract wound, theamelioration agent for the function of the digestive tract including theagent for recovery of the mucosal barrier function and the ameliorationagent for digestive and absorptive function. The ulcers includedigestive ulcers and erosions, acute ulcers, namely, acute mucosallesions.

[0197] The subject method, because of promoting proliferation ofintestinal mucosa, can be used in the treatment and prevention ofpathologic conditions of insufficiency in digestion and absorption, thatis, treatment and prevention of mucosal atrophy, or treatment ofhypoplasia of the digestive tract tissues and decrease in these tissuesby surgical removal as well as improvement of digestion and absorption.Further, the subject method can be used in the treatment of pathologicmucosal conditions due to inflammatory diseases such as enteritis,Crohn's disease and ulceric colitis and also in the treatment ofreduction in function of the digestive tract after operation, forexample, in damping syndrome as well as in the treatment of duodenalulcer in conjunction with the inhibition of peristalsis of the stomachand rapid migration of food from the stomach to the jejunum.Furthermore, glicentin can effectively be used in promoting cure ofsurgical invasion as well as in improving functions of the digestivetract. Thus, the present invention also provides a therapeutic agent foratrophy of the digestive tract mucosa, a therapeutic agent for wounds inthe digestive tract and a drug for improving functions of the digestivetract which comprise glicentin as active ingredients.

[0198] Likewise, the DPIV inhibitors of the subject invention can beused to alter the plasma half-life of secretin, VIP, PHI, PACAP, GIPand/or helodermin. Additionally, the subject method can be used to alterthe pharmacokinetics of Peptide YY and neuropeptide Y, both members ofthe pancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

[0199] Neuropeptide Y (NPY) is believed to act in the regulationvascular smooth muscle tone, as well as regulation of blood pressure.NPY also decreases cardiac contractility. NPY is also the most powerfulappetite stimulant known (Wilding et al., (1992) J Endocrinology132:299-302). The centrally evoked food intake (appetite stimulation)effect is predominantly mediated by NPY Y1 receptors and causes increasein body fat stores and obesity (Stanley et al., (1989) Physiology andBehavior 46:173-177).

[0200] According to the present invention, a method for treatment ofanorexia comprises administering to a host subject an effective amountof a DPIV inhibitor to stimulate the appetite and increase body fatstores which thereby substantially relieves the symptoms of anorexia.

[0201] A method for treatment of hypotension comprises administering toa host subject an effective amount of a DPIV inhibitor of the presentinvention to mediate vasoconstriction and increase blood pressure whichthereby substantially relieves the symptoms of hypotension.

[0202] DPIV has also been implicated in the metabolism and inactivationof growth hormone-releasing factor (GHRF). GHRF is a member of thefamily of homologous peptides that includes glucagon, secretin,vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI),pituitary adenylate cyclase activating peptide (PACAP), gastricinhibitory peptide (GIP) and helodermin. Kubiak et al. (1994) PeptideRes 7:153. GHRF is secreted by the hypothalamus, and stimulates therelease of growth hormone (GH) from the anterior pituitary. Thus, thesubject method can be used to improve clinical therapy for certaingrowth hormone deficient children, and in clinical therapy of adults toimprove nutrition and to alter body composition (muscle vs. fat). Thesubject method can also be used in veterinary practice, for example, todevelop higher yield milk production and higher yield, leaner livestock.

[0203] C. Examples of Peptidyl DPIV Inhibitors

[0204] In the case of DPIV inhibitors, a preferred class of inhibitorsare peptidyl compounds based on the dipeptides Pro-Pro or Ala-Pro.Another preferred class of peptidyl inhibitors are compounds based onthe dipeptide (D)-Ala-(L)-Ala. In many embodiments, it will be desirableto provide the peptidyl moiety as a peptidomimetic, e.g., to increasebioavailability and/or increase the serum half-life relative to theequivalent peptide. For instance, a variety of peptide backbone analogsare available in the art and be readily adpated for use in the subjectmethods.

[0205] In an exemplary embodiment, the peptidomimetic can be derived asa retro-inverso analog of the peptide. To illustrate, certain of thesubject peptides can be generated as the retro-inverso analog (shown inits unprotected state):

[0206] Such retro-inverso analogs can be made according to the methodsknown in the art, such as that described by the Sisto et al. U.S. Pat.No. 4,522,752. For example, the illustrated retro-inverso analog can begenerated as follows. The geminal diamine corresponding to theN-terminal amino acid analogs is synthesized by treating anN-Boc-protected amino acid (having the sidechain R) with ammonia underHOBT-DCC coupling conditions to yield amide, and then effecting aHofmann-type rearrangement with I,I-bis-(trifluoroacetoxy)iodobenzene(TIB), as described in Radhakrishna et al. (1979) J. Org. Chem. 44:1746.The product amine salt is then coupled to a side-chain protected (e.g.,as the benzyl ester) N-Fmoc D-enatiomer of the second amino acid residue(e.g., having a sidechain R′) under standard conditions to yield thepseudodipeptide. The Fmoc (fluorenylmethoxycarbonyl) group is removedwith piperidine in dimethylformamide, and the resulting amine istrimethylsilylated with bistrimethylsilylacetamide (BSA) beforecondensation with suitably alkylated, side-chain protected derivative ofMeldrum's acid, as described in U.S. Pat. No. 5,061,811 to Pinori etal., to yield the retro-inverso tripeptide analog. The pseudotripeptideis then coupled with (protected) boro-proline under standard conditionsto give the protected tetrapeptide analog. The protecting groups areremoved to release the final product, which is purified by HPLC.

[0207] In another illustrative embodiment, the peptidomimetic can bederived as a retro-enantio analog of the peptide.

[0208] Retro-enantio analogs such as this can be synthesized usingD-enatiomers of commercially available D-amino acids or other amino acidanalogs and standard solid- or solution-phase peptide-synthesistechniques.

[0209] In still another illustrative embodiment, trans-olefinderivatives can be made with the subject boronophenylalanine analogs.For example, an exemplary olefin analog is:

[0210] The trans olefin analog can be synthesized according to themethod of Y. K. Shue et al. (1987) Tetrahedron Letters 28:3225.

[0211] Still another class of peptidomimetic boronophenylalaninederivatives include the phosphonate derivatives, such as:

[0212] The synthesis of such phosphonate derivatives can be adapted fromknown synthesis schemes. See, for example, Loots et al. in Peptides:Chemistry and Biology, (Escom Science Publishers, Leiden, 1988, p. 118);Petrillo et al. in Peptides: Structure and Function (Proceedings of the9th American Peptide Symposium, Pierce Chemical Co. Rockland, Ill.,1985).

[0213] D. Non-Peptidyl DPIV Inhibitors

[0214] The pharmaceutical industry has developed a variety of differentstrategies for assessing millions of compounds a year as potential leadcompounds based on inhibitory activity against an enzyme. DPIV and otherproteolytic enzymes targeted by the subject method are amenable to thetypes of high throughput screening required to sample large arrays ofcompounds and natural extracts for suitable inhibitors.

[0215] As an illustrative embodiment, the ability of a test agent toinhibit DPIV can be assessed using a colorimetric or fluorometricsubstrate, such as Ala-Pro-paranitroanilide. See U.S. Pat. No.5,462,928. Moreover, DPIV can be purified, and is accordingly readilyamenable for use in such high throughput formats as multi-well plates.

[0216] Briefly, DPIV is purified from pig kidney cortex (Barth et al.(1974) Acta Biol Med Germ 32:157; Wolf et al. (1972) Acta Bio Mes Germ37:409) or human placenta (Puschel et al. (1982) Eur J Biochem 126:359).An illustrative reaction mixture includes 50 μM sodium Hepes (pH7.8), 10μM Ala-Pro-paranitroanilide, 6 milliunits of DPIV, and 2% (v/v)dimethylformamide in a total volume of 1.0 mL. The reaction is initiatedby addition of enzyme, and formation of reaction product(paranitroanilide) in the presence and absence of a test compound can bedetected photometrically, e.g., at 410 nm.

[0217] Exemplary compounds which can be screened for activity againstDPIV (or other relevant enzymes) include peptides, nucleic acids,carbohydrates, small organic molecules, and natural product extractlibraries, such as isolated from animals, plants, fungus and/ormicrobes.

[0218] E. Assays of Insulinotropic Activity

[0219] In selecting a compound suitable for use in the subject method,it is noted that the insulinotropic property of a compound may bedetermined by providing that compound to animal cells, or injecting thatcompound into animals and monitoring the release of immunoreactiveinsulin (IRI) into the media or circulatory system of the animal,respectively. The presence of IRI can be detected through the use of aradioimmunoassay which can specifically detect insulin.

[0220] The db/db mouse is a genetically obese and diabetic strain ofmouse. The db/db mouse develops hyperglycemia and hyperinsulinemiaconcomitant with its development of obesity and thus serves as a modelof obese type 2 diabetes (NIDDM). The db/db mice can purchased from, forexample, The Jackson Laboratories (Bar Harbor, Me.). In an exemplaryembodiment, for treatment of the mice with a regimen including a DPIVinhibitor or control, sub-orbital sinus blood samples are taken beforeand at some time (e.g., 60 minutes) after dosing of each animal. Bloodglucose measurements can be made by any of several conventionaltechniques, such as using a glucose meter. The blood glucose levels ofthe control and DPIV inhibitor dosed animals are compared

[0221] The metabolic fate of exogenous GLP-1 can also be followed ineither nondiabetic and type II diabetic subjects, and the effect of acandidate DPIV inhibitor determined. For instance, a combination ofhigh-pressure liquid chromatography (HPLC), specific radioimmunoassays(RIAs), and a enzyme-linked immunosorbent assay (ELISA), can be used,whereby intact biologically active GLP-1 and its metabolites can bedetected. See, for example, Deacon et al. (1995) Diabetes 44:1126-1131,.To illustrate, after GLP-1 administration, the intact peptide can bemeasured using an NH2-terminally directed RIA or ELISA, while thedifference in concentration between these assays and aCOOH-terminal-specific RIA allowed determination of NH2-terminallytruncated metabolites. Without inhibitor, subcutaneous GLP-1 is rapidlydegraded in a time-dependent manner, forming a metabolite whichco-elutes on HPLC with GLP-I(9-36) amide and has the same immunoreactiveprofile. For instance, thirty minutes after subcutaneous GLP-1administration to diabetic patients (n=8), the metabolite-accounted for88.5+1.9% of the increase in plasma immunoreactivity determined by theCOOH-terminal RIA, which was higher than the levels measured in healthysubjects (78.4+3.2%; n=8; P<0.05). See Deacon et al., supra.Intravenously infused GLP-I was also extensively degraded.

[0222] F. Pharmaceutical Formulations The inhibitors can be administeredin various forms, depending on the disorder to be treated and the age,condition and body weight of the patient, as is well known in the art.For example, where the compounds are to be administered orally, they maybe formulated as tablets, capsules, granules, powders or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular or subcutaneous), drop infusion preparationsor suppositories. For application by the ophthalmic mucous membraneroute, they may be formulated as eyedrops or eye ointments. Theseformulations can be prepared by conventional means, and, if desired, theactive ingredient may be mixed with any conventional additive, such asan excipient, a binder, a disintegrating agent, a lubricant, acorrigent, a solubilizing agent, a suspension aid, an emulsifying agentor a coating agent. Although the dosage will vary depending on thesymptoms, age and body weight of the patient, the nature and severity ofthe disorder to be treated or prevented, the route of administration andthe form of the drug, in general, a daily dosage of from 0.01 to 2000 mgof the compound is recommended for an adult human patient, and this maybe administered in a single dose or in divided doses.

[0223] Glucose metabolism can be altered, and symptoms associated withtype II diabetes can be decreased or eliminated, in accordance with a“timed” administration of DPIV inhibitors wherein one or moreappropriate indices for glucose metabolism and/or type II diabetes canbe used to assess effectiveness of the treatment (dosage and/or timing):e.g. glucose tolerance, glucose level, insulin level, insulinsensitivity, glycosylated hemoglobin.

[0224] An effective time for administering DPIV inhibitors needs to beidentified. This can be accomplished by routing experiment as describedbelow, using one or more groups of animals (preferably at least 5animals per group).

[0225] In animals, insulinotropic activity by DPIV inhibitor treatmentcan be assessed by administering the inhibitor at a particular time ofday and measuring the effect of the administration (if any) by measuringone or more indices associated with glucose metabolism, and comparingthe post-treatment values of these indices to the values of the sameindices prior to treatment.

[0226] The precise time of administration and/or amount of DPIVinhibitor that will yield the most effective results in terms ofefficacy of treatment in a given patient will depend upon the activity,pharmacokinetics, and bioavailability of a particular compound,physiological condition of the patient (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosageand type of medication), route of administration, etc. However, theabove guidelines can be used as the basis for fine-tuning the treatment,e.g., determining the optimum time and/or amount of administration,which will require no more than routine experimentation consisting ofmonitoring the subject and adjusting the dosage and/or timing.

[0227] While the subject is being treated, glucose metabolism ismonitored by measuring one or more of the relevant indices atpredetermined times during a 24-hour period. Treatment (amounts, timesof administration and type of medication) may be adjusted (optimized)according to the results of such monitoring. The patient is periodicallyreevaluated to determine extent of improvement by measuring the sameparameters, the first such reevaluation typically occurring at the endof four weeks from the onset of therapy, and subsequent reevaluationsoccurring every 4 to 8 weeks during therapy and then every 3 monthsthereafter. Therapy may continue for several months or even years withsix months being a typical length of therapy for humans.

[0228] Adjustments to the amount(s) of drug(s) administered and possiblyto the time of administration may be made based on these reevaluations.For example, if after 4 weeks of treatment one of the metabolic indiceshas not improved but at least one other one has, the dose could beincreased by ⅓ without changing the time of administration.

[0229] Treatment can be initiated with smaller dosages which are lessthan the optimum dose of the compound. Thereafter, the dosage should beincreased by small increments until the optimum effect under thecircumstances is reached. For convenience, the total daily dosage may bedivided and administered in portions during the day if desired.

[0230] The phrase “therapeutically-effective amount” as used hereinmeans that amount of, e.g., a DPIV inhibitor(s), which is effective forproducing some desired therapeutic effect by inhibiting, for example,the proteolysis of a peptide hormone at a reasonable benefit/risk ratioapplicable to any medical treatment.

[0231] The phrase “pharmaceutically acceptable” is employed herein torefer to those DPIV inhibitors, materials, compositions, and/or dosageforms which are, within the scope of sound medical judgment, suitablefor use in contact with the tissues of human beings and animals withoutexcessive toxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

[0232] The phrase “pharmaceutically-acceptable carrier” as used hereinmeans a pharmaceutically-acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting the subjectchemical from one organ, or portion of the body, to another organ, orportion of the body. Each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid, (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

[0233] The term “pharmaceutically-acceptable salts” refers to therelatively non-toxic, inorganic and organic acid addition salts of DPIVinhibitors. These salts can be prepared in situ during the finalisolation and purification of the DPIV Inhibitors, or by separately.reacting a purified DPIV inhibitor in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

[0234] In other cases, the DPIV inhibitor useful in the methods of thepresent invention may contain one or more acidic functional groups and,thus, are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of a DPIV inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the DPIV inhibitor(s), or by separately reacting the purified DPIVinhibitor(s) in its free acid form with a suitable base, such as thehydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

[0235] Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

[0236] Examples of pharmaceutically-acceptable antioxidants include: (1)water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0237] Formulations useful in the methods of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred per cent, this amount will range from about 1 per cent to aboutninety-nine percent of active ingredient, preferably from about 5 percent to about 70 per cent, most preferably from about 10 per cent toabout 30 per cent.

[0238] Methods of preparing these formulations or compositions includethe step of bringing into association a DPIV inhibitor(s) with thecarrier and, optionally, one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing intoassociation a DPIV inhibitor with liquid carriers, or finely dividedsolid carriers, or both, and then, if necessary, shaping the product.

[0239] Formulations suitable for oral administration may be in the formof capsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a DPIV inhibitor(s) as an active ingredient. Acompound may also be administered as a bolus, electuary or paste.

[0240] In solid dosage forms for oral administration (capsules, tablets,pills, dragees, powders, granules and the like), the active ingredientis mixed with one or more pharmaceutically-acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

[0241] A tablet may be made by compression or molding, optionally withone or more accessory ingredients. Compressed tablets may be preparedusing binder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

[0242] Tablets, and other solid dosage forms, such as dragees, capsules,pills and granules, may optionally be scored or prepared with coatingsand shells, such as enteric coatings and other coatings well known inthe pharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These. compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

[0243] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active ingredient,the liquid dosage forms may contain inert diluents commonly used in theart, such as, for example, water or other solvents, solubilizing agentsand emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof.

[0244] Besides inert diluents, the oral compositions can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, coloring, perfuming and preservative agents.

[0245] Suspensions, in addition to the active DPIV inhibitor(s) maycontain suspending agents as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

[0246] Formulations for rectal or vaginal administration may bepresented as a suppository, which may be prepared by mixing one or moreDPIV inhibitor(s) with one or more suitable nonirritating excipients orcarriers comprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

[0247] Formulations which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

[0248] Dosage forms for the topical or transdermal administration of aDPIV inhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

[0249] The ointments, pastes, creams and gels may contain, in additionto DPIV inhibitor(s), excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

[0250] Powders and sprays can contain, in addition to a DPIVinhibitor(s), excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

[0251] The DPIV inhibitor(s) can be alternatively administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension could be used. Sonicnebulizers are preferred because they minimize exposing the agent toshear, which can result in degradation of the compound.

[0252] Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

[0253] Transdermal patches have the added advantage of providingcontrolled delivery of a DPIV inhibitor(s) to the body. Such dosageforms can be made by dissolving or dispersing the agent in the propermedium. Absorption enhancers can also be used to increase the flux ofthe peptidomimetic across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe peptidomimetic in a polymer matrix or gel.

[0254] Ophthalmic formulations, eye ointments, powders, solutions andthe like, are also contemplated as being within the scope of thisinvention.

[0255] Pharmaceutical compositions of this invention suitable forparenteral administration comprise one or more DPIV inhibitor(s) incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

[0256] Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

[0257] These compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

[0258] In some cases, in order to prolong the effect of a drug, it isdesirable to slow the absorption of the drug from subcutaneous orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material having poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle.

[0259] Injectable depot forms are made by forming microencapsulematrices of DPIV inhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

[0260] When the DPIV inhibitor(s) of the present invention areadministered as pharmaceuticals, to humans and animals, they can begiven per se or as a pharmaceutical composition containing, for example,0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

[0261] The preparations of agents may be given orally, parenterally,topically, or rectally. They are of course given by forms suitable foreach administration route. For example, they are administered in tabletsor capsule form, by injection, inhalation, eye lotion, ointment,suppository, etc. administration by injection, infusion or inhalation;topical by lotion or ointment; and rectal by suppositories. Oraladministration is preferred.

[0262] The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

[0263] The phrases “systemic administration,” “administeredsystemically,”, “peripheral administration” and “administeredperipherally” as used herein mean the administration of a DPIVinhibitor, drug or other material other than directly into the centralnervous system, such that it enters the patient's system and, thus, issubject to metabolism and other like processes, for example,subcutaneous administration.

[0264] These DPIV inhibitor(s) may be administered to humans and otheranimals for therapy by any suitable route of administration, includingorally, nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

[0265] Regardless of the route of administration selected, the DPIVinhibitor(s), which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

[0266] Actual dosage levels of the active ingredients in thepharmaceutical compositions of this invention may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

[0267] G. Conjoint Administration

[0268] Another aspect of the invention provides a conjoint therapywherein one or more other therapeutic agents are administered with theprotease inhibitor. Such conjoint treatment may be achieved by way-ofthe simultaneous, sequential or separate dosing of the individualcomponents of the treatment.

[0269] In one embodiment, a DPIV inhibitor is conjointly administeredwith insulin or other insulinotropic agents, such as GLP-1 or a genetherapy vector which causes the ectopic expression of GLP-1.

[0270] In another illustrative embodiment, the subject inhibitors can beconjointly administered with a an M1 receptor antagonist. Cholinergicagents are potent modulators of insulin release that act via muscarinicreceptors. Moreover, the use of such agents can have the added benefitof decreasing cholesterol levels, while increasing HDL levels. Suitablemuscarinic receptor antagonists include substances that directly orindirectly block activation of muscarinic cholinergic receptors.Preferably, such substances are selective (or are used in amounts thatpromote such selectivity) for the M1 receptor. Nonlimiting examplesinclude quaternary. amines (such as methantheline, ipratropium, andpropantheline), tertiary amines (e.g. dicyclomine, scopolamine) andtricyclic amines (e.g. telenzepine). Pirenzepine and methyl scopolamineare preferred. Other suitable muscarinic receptor antagonists includebenztropine (conmmercially available as COGENTINfrom Merck),hexahydro-sila-difenidol hydrochloride (HHSID hydrochloride disclosed inLambrecht et al. (1989) Trends in Pharrnacol. Sci. 10(Suppl):60;(+/−)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate(QNX-hemioxalate; Birdsall et al., Trends in Pharmacol. Sci. 4:459,1983; telenzepine dihydrochloride (Coruzzi et al. (1989) Arch. Int.Pharmacodyn. Ther. 302:232; and Kawashima et al. (1990) Gen. Pharmacol.21:17) and atropine. The dosages of such muscarinic receptor antagonistswill be generally subject to optimization as outlined above. In the caseof lipid metabolism disorders, dosage optimization may be necessaryindependently of whether administration is timed by reference to thelipid metabolism responsiveness window or not.

[0271] In terms of regulating insulin and lipid metabolism and reducingthe foregoing disorders, the subject DPIV inhibitors may also actsynergistically with prolactin inhibitors such as d2 dopamine agonists(e.g. bromocriptine). Accordingly, the subject method can include theconjoint administration of such prolactin inhibitors asprolactin-inhibiting ergo alkaloids and prolactin-inhibiting dopamineagonists. Examples of suitable compounds include2-bromo-alpha-ergocriptine, 6-methyl-8beta-carbobenzyloxyaminoethyl-10-alpha-ergoline, 8-acylaminoergolines,6-methyl-8-alpha-(N-acyl)amino-9-ergoline,6-methyl-8-alpha-(N-phenylacetyl)amino-9-ergoline, ergocomine,9,10-dihydroergocornine, D-2-halo-6-alkyl-8-substituted ergolines,D-2-bromo-6-methyl-8-cyanomethylergoline, carbidopa, benserazide andother dopadecarboxylase inhibitors, L-dopa, dopamine and non toxic saltsthereof.

[0272] The DPIV inhibitors used according to the invention can also beused conjointly with agents acting on the ATP-dependent potassiumchannel of the β-cells, such as glibenclamide, glipizide, gliclazide andAG-EE 623 ZW. The DPIV inhibitors may also advantageously be applied incombination with other oral agents such as metformin and relatedcompounds or glucosidase inhibitors as, for example, acarbose.

EXEMPLIFICATION

[0273] The invention now being generally described, it will be morereadily understood by reference to the following examples which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Synthesis of BoroProline

[0274] Referring to FIG. 1, the starting compound I is preparedessentially by the procedure of Matteson et al. (Organometallics 3:1284,1984), except that a pinacol ester is substituted for the pinanediolester. Similar compounds such as boropipecolic acid and 2-azetodineboronic acid can be prepared by making the appropriate selection ofstarting material to yield the pentyl and propyl analogs of compound I.Further, C1 can be substituted for Br in the formula, and other diolprotecting groups can be substituted for pinacol in the formula, e.g.,2,3-butanediol and alphapinanediol.

[0275] Compound II is prepared by reacting compound I with[(CH₃)O₃Si]₂N-Li⁺. In this reaction hexamethyldisilazane is dissolved intetrahydrofuran and an equivalent of n-butyllithium added at −78° C.After warming to room temperature (20° C.) and cooling to −78° C., anequivalent of compound I is added in tetrahydrofuran. The mixture isallowed to slowly come to room temperature and to stir overnight. Thealpha-bis[trimethylsilane]-protected amine is isolated by evaporatingsolvent and adding hexane under anhydrous conditions. Insoluble residueis removed by filtration under a nitrogen blanket, yielding a hexanesolution of compound II.

[0276] Compound III, the N-trimethysilyl protected form of boroProlineis obtained by the thermal cyclization of compound II during thedistillation process in which compound II is heated to 100-150° C. anddistillate is collected which boils 66-62° C. at 0.06-0.10 mm pressure.

[0277] Compound IV, boroProline-pinacol hydrogen chloride, is obtainedby treatment of compound III with HCl:dioxane. Excess HCl andby-products are removed by trituration with ether. The final product isobtained in a high degree of purity by recrystallization from ethylacetate.

[0278] The boroProline esters can also be obtained by treatment of thereaction mixture obtained in the preparation of compound II withanhydrous acid to yield 1-amino-4-bromobutyl boronate pinacol as a salt.Cyclization occurs after neutralizing the salt with base and heating thereaction.

Example 2 Preparation of boroProline-Pinacol

[0279] The intermediate, 4-Bromo-1-chlorobutyl boronate pinacol, wasprepared by the method in Matteson et al. (Organometallics 3:1284, 1984)except that conditions were modified for large scale preparations andpinacol was substituted for the pinanediol protecting group.

[0280] 3-bromopropyl boronate pinacol was prepared by hydrogenboronationof allyl bromide (173 ml, 2.00 moles) with catechol borane (240 ml, 2.00moles). Catechol borane was added to allyl bromide and the reactionheated for 4 hours at 100° C. under a nitrogen atmosphere. The product,3-bromopropyl boronate catechol (bp 95-102° C., 0.25 mm), was isolatedin a yield of 49% by distillation. The catechol ester (124 g, 0.52moles) was transesterified with pinacol (61.5 g, 0.52 moles) by mixingthe component in 50 ml of THF and allowing them to stir for 0.5 hours at0° C. and 0.5 hours at room temperature. Solvent was removed byevaporation and 250 ml of hexane added. Catechol was removed as acrystalline solid. Quantitative removal was achieved by successivedilution to 500 ml and to 1000 ml with hexane and removing crystals ateach dilution. Hexane was evaporated and the product distilled to yield177 g (bp 60-64° C., 0.35 mm).

[0281] 4-Bromo-1-chlorobutyl boronate pinacol was prepared byhomologation of the corresponding propyl boronate. Methylene chloride(50.54 ml, 0.713 moles) was dissolved in 500 ml of THF, 1.54Nn-butyllithium in hexane (480 ml, 0.780 moles) was slowly added at −100°C. 3-Bromopropyl boronate pinacol (178 g, 0.713 moles) was dissolved in500 ml of THG, cooled to the freezing point of the solution, and addedto the reaction mixture. Zinc chloride (54.4 g, 0.392 moles) wasdissolved in 250 ml of THG, cooled to 0° C., and added to the reactionmixture in several portions. The reaction was allowed to slowly warm toroom temperature and to stir overnight. Solvent was evaporated and theresidue dissolved in hexane (1 liter) and washed with water (1 liter).Insoluble material was discarded. After drying over anhydrous magnesiumsulfate and filtering, solvent was evaporated. The product was distilledto yield 147 g (bp 110-112° C., 0.200 mm).

[0282] N-Trimethylsilyl-boroProline pinacol was prepared first bydissolving hexamethyldisilizane (20.0 g, 80.0 mmoles) in 30 ml of THF,cooling the solution to −78° C., and adding 1.62N n-butyllithium inhexane (49.4 ml, 80.0 mmoles). The solution was allowed to slowly warmto room temperature. It was recooled to −78° C. and4-bromo-1-chlorobutyl boronate pinacol (23.9 g, 80.0 mmoles) added in 20ml of THF. The mixture was allowed to slowly warm to room temperatureand to stir overnight. Solvent was removed by evaporation and dry hexane(400 ml) added to yield a precipitant which was removed by filbrationunder a nitrogen atmosphere. The filtrate was evaporated and the residuedistilled, yielding 19.4 g of the desired product (bp 60-62° C.,0.1-0.06 mm).

[0283] H-boroProline-pinacol HCl (boroProline-pinacol. HCl) was preparedby cooling N-trimrethylsilyl-boroProline pinacol (16.0 g, 61.7 mmoles)to −78° C. and adding 4N HCl:dioxane 46 ml, 185 mmoles). The mixture wasstirred 30 minutes at −78° C. and 1 hour at room temperature. Solventwas evaporated and the residue triturated with ether to yield a solid.The crude product was dissolved in chloroform and insoluble materialremoved by filtration. The solution was evaporated and the productcrystallized from ethyl acetate to yield 11.1 g of the desired product(mp 156.5-157° C.).

Example 3 Synthesis of BoroProline Peptides

[0284] General methods of coupling of N-protected peptides and aminoacids with suitable side-chain protecting groups toH-boroProline-pinacol are applicable. When needed, side-chain protectingand N-terminal protecting groups can be removed by treatment withanhydrous HCl, HBr, trifluoroacetic acid, or by catalytic hydrogenation.These procedures are known to those skilled in the art of peptidesynthesis.

[0285] The mixed anhydride procedure of Anderson et al. (J. Am. Chem.Soc. 89:5012, 1984) is preferred for peptide coupling. Referring againto FIG. 1, the mixed anhydride of an N-protected amino acid or a peptideis prepared by dissolving the peptide in tetrahydrofuran and adding oneequivalent of N-methylmorpholine. The solution is cooled to −20° C. andan equivalent of isobutyl chloroformate is added. After 5 minutes, thismixture and one equivalent of triethylamine (or other stericallyhindered base) are added to a solution of H-boroPro-pinacol dissolved ineither cold chloroform of tetrahydrofuran.

[0286] The reaction mixture is routinely stirred for one hour at −20° C.and 1 to 2 hours at room temperature (20° C.). Solvent is removed byevaporation, and the residue is dissolved in ethyl acetate. The organicsolution is washed with 0.20N hydrochloric acid, 5% aqueous sodiumbicarbonate, and saturated aqueous sodium chloride. The organic phase isdried over anhydrous sodium sulfate, filtered, and evaporated. Productsare purified by either silica gel chromatography or gel permeationchromatography using Sephadex TM LH-20 and methanol as a solvent.

[0287] Previous studies have shown that the pinacol protecting group canbe removed in situ by preincubation in phosphate buffer prior to runningbiological experiments (Kettner et al., J. Biol. Chem. 259:15106, 1984).Several other methods are also applicable for removing pinacol groupsfrom peptides, including boroProline, and characterizing the finalproduct. First, the peptide can be treated with diethanolamine to yieldthe corresponding diethanolamine boronic acid ester, which can bereadily hydrolyzed by treatment with aqueous acid or a sulfonic acidsubstituted polystyrene resin as described in Kettner et al. (supra).Both pinacol and pinanediol protecting groups can be removed by treatingwith BC13 in methylene chloride as described by Kinder et al. (J. Med.Chem. 28:1917). Finally, the free boronic acid can be converted to thedifluoroboron derivative (-BF2) by treatment with aqueous HF asdescribed by Kinder et al. (supra).

[0288] Similarly, different ester groups can be introduced by reactingthe free boronic acid with various di-hydroxy compounds (for example,those containing heteroatoms such as S or N) in an inert solvent.

Example 4 Preparation of H-Ala-BoroPro

[0289] Boc-Ala-boroPro was prepared by mixed anhydride coupling of theN-Boc-protected alanine and H-boroPro prepared as described above.H-Ala-boroPro (Ala-boroPro) was prepared by removal of the Bocprotecting group at 0° C. in 3.5 molar excess of 4N HCl-dioxane. Thecoupling and deblocking reactions were performed by standard chemicalreaction. Ala-boroPro has a K_(i) for-DP-IV of in the nanomolar range.Boc-blocked Ala-boroPro has no affinity for DP-IV.

[0290] The two diastereomers of Ala-boroPro-pinacol,L-Ala-D-boroPro-pinacol and L-Ala-L-boroPro-pinacol, can be partiallyseparated by silica gel chromatography with 20% methanol in ethylacetate as eluant. The early fraction appears by NMR analysis to be 95%enriched in one isomer. Because this fraction has more inhibits DP-IV toa greater extent than later fractions (at equal concentrations) it isprobably enriched in the L-boroPro (L-Ala-L-boroPro-pinacol) isomer.

Example 5 Glucose Tolerance Test

[0291] Experiments show that Pro-boro-pro clearly lowers blood sugarbased upon results from an oral glucose challenge in mice. The first twoexperiments are “acute” experiments wherein the mice were injected witha single dose of Pro-boro-pro. In the first set of experiments mice wereinjected with 150 μg of Pro-boro-pro (PBP-1) and then subjected to anoral glucose tolerance test within an hour. 8 μg of GLP-1 was alsoadministered to these mice five minutes prior to administration ofglucose. See FIG. 2. In a second set of experiments mice were injectedwith Pro-boro-pro (PBP-2) one hour prior to an oral glucose challengetest. FIG. 3 presents the results of these experiments. Each set ofexperiments was also performed using saline as a control.

[0292] The third set of experiments were “chronic” experiments, whereinthe mice were injected twice daily with Pro-boro-pro for four days,followed by an oral glucose challenge. These results are presented inFIG. 4.

Example 6 Glucose Tolerance Test, Comparison of Normal and GLP-1Receptor −/− Mice

[0293] GLP-1 receptor gene “knock-out” causes glucose intolerance intransgenic mice. Gallwitz B; Schmidt WE Z Gastroenterol (1997) 35:655-8. FIG. 5 compares the ability of Pro-boro-pro to lower plasmaglucose levels in normal and GLP-1 receptor −/− transgenic mice.

[0294] All of the above-cited references and publications are herebyincorporated by reference.

Equivalents

[0295] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. A method for modifying, in an animal, metabolism ofglucagon-like peptide 1 (GLP-1), comprising administering to the animala composition including one or more inhibitors of a dipeptidylpeptidasewhich inactivates GLP-1, which inhibitor(s) are administered in anamount sufficient to inhibit the dipeptidylpeptidase proteolysis ofGLP-1.
 2. A method for modifying glucose metabolism of an animal,comprising administering to the animal a composition including one ormore protease inhibitors which inhibit DPIV-mediated proteolysis with aKi of 1 nM or less.
 3. A method for modifying glucose metabolism of ananimal, comprising administering to the animal a composition includingone or more protease inhibitors which inhibit the proteolysis ofglucagon-like peptide 1 (GLP-1) and accordingly increase the plasmahalf-life of GLP-1.
 4. A method for treating Type II diabetes,comprising administering to an animal a composition including one ormore inhibitors dipeptidylpeptidase IV (DPIV).
 5. The method of claim 1,wherein dipeptidylpeptidase is DPIV.
 6. The method of claim 3, whereinprotease inhibitor is an inhibitor of DPIV.
 7. The method of claim 2 or3, wherein administering the inhibitor reduces one or more of insulinresistance, glucose intolerance, hyperglycemia, hyperinsulinemia,obesity, hyperlipidemia, hyperlipoproteinemia.
 8. The method of claim 1,2, 3 or 4, wherein the inhibitor has an EC50 for modification of glucosemetabolism which is at least one order of magnitude less than its EC50for immunosuppression.
 9. The method of claim 1, 2, 3 or 4, wherein theinhibitor has an EC50 for inhibition of glucose tolerance in thenanomolar or less range
 10. The method of claim 1, 2, 3 or 4, whereinthe inhibitor has an EC50 for immunosuppression in the μM or greaterrange.
 11. The method of claim 4, 5 or 6, wherein the inhibitor has a Kifor DPIV inhibition of 1.0 nm or less.
 12. The method of claim 1, 2, 3or 4, wherein the inhibitor is peptidomimetic of a peptide selected fromthe group consisting Pro-Pro, Ala-Pro, and (D)-Ala-(L)-Ala.
 13. Themethod of claim 1, 2, 3 or 4, wherein the inhibitor has a molecularweights less than 7500 amu.
 14. The method of claim 1, 2, 3 or 4,wherein the inhibitor is orally active.
 15. The method of claim 1, 2, 3or 4, wherein the inhibitor is represented by the general formula;

wherein A represents a 4-8 membered heterocycle including the N and theCα carbon; Z represents C or N; W represents a functional group whichreacts with an active site residue of the targeted protease, R₁represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

R₂ is absent or represents one or more substitutions to the ring A, eachof which can independently be a halogen, a lower alkyl, a lower alkenyl,a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, ora ketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an amino, an acylamino, an amido, a cyano, a nitro, anazido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₇,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇; if X is N, R₃represents hydrogen, if X is C, R₃ represents hydrogen, or a halogen, alower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as acarboxyl, an ester, a formate, or a ketone), a thiocarbonyl (such as athioester, a thioacetate, or a thiofomate), an amino, an acylamino, anamido, a cyano, a nitro, an azido, a sulfate, a sulfonate, asulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇; R₆ represents hydrogen, a halogen, a alkyl, aalkenyl, a alkynyl, an aryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH,—(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl,—(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl,—(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,R₇ represents, for each occurrence, a substituted or unsubstituted aryl,aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; m is zero or aninteger in the range of 1 to 8; and n is an integer in the range of 1 to8.
 16. The method of claim 15, wherein W represents —CN, —CH═NR₅,

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m—R₇, —(CH₂)n—OH, —(CH₂)n—O-alkyl, —(CH₂)n—O-alkenyl,—(CH₂)n—O-alkynyl, —(CH₂)n—O—(CH₂)m—R₇, —(CH₂)n—SH, —(CH₂)n—S-alkyl,—(CH₂)n—S-alkenyl, —(CH₂)n—S-alkynyl, —(CH₂)n—S—(CH₂)m—R₇, —C(O)C(O)NH₂,—C(O)C(O)OR′₇; R′₇ represents, for each occurrence, hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; and Y₁ and Y₂ can independently ortogether be OH, or a group capable of being hydrolyzed to a hydroxylgroup, including cyclic derivatives where Y₁ and Y₂ are connected via aring having from 5 to 8 atoms in the ring structure (such as pinacol orthe like), R₅₀ represents O or S; R₅₁ represents N₃, SH₂, NH₂, NO₂ orOR′₇; R₅₂ represents hydrogen, a lower alkyl, an amine, OR′₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure X1 represents a halogen;X₂ and X₃ each represent a hydrogen or a halogen
 18. The method of claim16, wherein the ring A is represented by the formula

wherein n is an integer of 1 or
 2. 19. The method of claim 16, wherein Wrepresents


20. The method of claim 16, wherein R1 represents

wherein R36 is a small hydrophobic group and R38 is hydrogen, or, R36and R38 together form a 4-7 membered heterocycle including the N and theCα carbon, as defined for A above; and R40 represents a C-terminallylinked amino acid residue or amino acid analog, or a C-terminally linkedpeptide or peptide analog, or an amino-protecting group
 21. The methodof claim 16, wherein R2 is absent, or represents a small hydrophobicgroup.
 22. The method of claim 16, wherein R3 is a hydrogen, or a smallhydrophobic group.
 23. The method of claim 16, wherein R5 is a hydrogen,or a halogentated lower alkyl.
 24. The method of claim 16, wherein X1 isa fluorine, and X2 and X3, if halogens, are fluorine.
 25. The method ofclaim 16, wherein the inhibitor is represented by the general formula:

wherein R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a terminally linked peptide or peptide analog, or

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇, or R₈ and R₉ taken together with the N atom towhich they are attached complete a heterocyclic ring having from 4 to 8atoms in the ring structure; R₁₁ and R₁₂ each independently representhydrogen, a alkyl, or a pharmaceutically acceptable salt, or R₁₁ and R₁₂taken together with the O—B—O atoms to which they are attached completea heterocyclic ring having from 5 to 8 atoms in the ring structure; m iszero or an integer in the range of 1 to 8; and n is an integer in therange of 1 to
 8. 26. The method of claim 16, wherein the inhibitor isrepresented by the general formula

wherein R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a terminally linked peptide or peptide analog, or

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)₂—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇, or R₈ and R₉ taken together with the N atom towhich they are attached complete a heterocyclic ring having from 4 to 8atoms in the ring structure; and m is zero or an integer in the range of1 to 8; and n is an integer in the range of 1 to
 8. 27. The method ofclaim 16 ,wherein the inhibitor is represented by the general formula:

wherein R₁ represents a C-terminally linked amino acid residue or aminoacid analog, or a terminally linked peptide or peptide analog, or

R₆ represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, anaryl, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl,—(CH₂)_(m)—O-alkenyl, —(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇,—(CH₂)_(m)—SH, —(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl,—(CH₂)_(m)—S-alkynyl, —(CH₂)_(m)—S—(CH₂)_(m)—R₇,

R₇ represents an aryl, a cycloalkyl, a cycloalkenyl, or a heterocycle;R₈ and R₉ each independently represent hydrogen, alkyl, alkenyl,—(CH₂)_(m)—R₇, —C(═O)-alkyl, —C(═O)-alkenyl, —C(═O)-alkynyl,—C(═O)—(CH₂)_(m)—R₇, or R₈ and R₉ taken together with the N atom towhich they are attached complete a heterocyclic ring having from 4 to 8atoms in the ring structure; X₁, X₂ and X₃ each represent a hydrogen ora halogen; and m is zero or an integer in the range of 1 to 8; and n isan integer in the range of 1 to
 8. 28. The method of claim 16 whereinthe inhibitor is represented by the general formula:

wherein R32 is a small hydrophobic group; and R30 represents aC-terminally linked amino acid residue or amino acid analog, or aC-terminally linked peptide or peptide analog, or an amino-protectinggroup.
 29. The method of claim 16 ,wherein the inhibitor is representedby the general formula

wherein W represents a functional group which reacts with an active siteresidue of the targeted protease, as for example, —CN, —CH═NR₅,

R₁ represents a C-terminally linked amino acid residue or amino acidanalog, or a C-terminally linked peptide or peptide analog, or anamino-protecting group, or

R₃ represents hydrogen or a halogen, a lower alkyl, a lower alkenyl, alower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, or aketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an amino, an acylamino, an amido, a cyano, a nitro, anazido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₇,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₇; R₅ represents H,an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃, —(CH₂)m—R₇, —(CH₂)n—OH,—(CH₂)n—O-alkyl, —(CH₂)n—O-alkenyl, —(CH₂)n—O-alkynyl,—(CH₂)n—O—(CH₂)m—R₇, —(CH₂)n—SH, —(CH₂)n—S-alkyl, —(CH₂)n—S-alkenyl,—(CH₂)n—S-alkynyl, —(CH₂)n—S—(CH₂)m—R₇, —C(O)C(O)NH₂, —C(O)C(O)OR′₇; R₆represents hydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, an aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R₇, R₇ represents, for each occurrence, asubstituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, orheterocycle; R′₇ represents, for each occurrence, hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; R₆₁ and R₆₂, independently, representsmall hydrophobic groups; Y₁ and Y₂ can independently or together be OH,or a group capable of being hydrolyzed to a hydroxyl group, includingcyclic derivatives where Y₁ and Y₂ are connected via a ring having from5 to 8 atoms in the ring structure (such as pinacol or the like), R₅₀represents O or S; R₅₁ represents N₃, SH₂, NH₂, NO₂ or OR′₇; R₅₂represents hydrogen, a lower alkyl, an amine, OR′7, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure X₁ represents a halogen;X₂ and X₃ each represent a hydrogen or a halogen m is zero or an integerin the range of 1 to 8; and n is an integer in the range of 1 to
 8. 30.A method for modifiying, in an animal, metabolism of peptide hormone,comprising administering to the animal a composition including one ormore inhibitors of dipeptidylpeptidase IV (DPIV) in an amount sufficientto increase the plasma half-life of a peptide hormone, which peptidehormone is selected from the group consisting of glucagon-like peptide 2(GLP-2), growth hormone-releasing factor (GHRF), vasoactive intestinalpeptide (VIP), peptide histidine isoleucine (PHI), pituitary adenylatecyclase activating peptide (PACAP), gastric inhibitory peptide (GIP),helodermin, Peptide YY and neuropeptide Y.
 31. A method for modifyingglucose metabolism of an animal, comprising administering to the animala composition including boronyl peptidomimetic of a peptide selectedfrom the group consisting Pro-Pro, Ala-Pro, and (D)-Ala-(L)-Ala.
 32. Themethod of claim 31, wherien the boronyl peptidomimetic is represented inthe general formula:

wherein each A independently represents a 4-8 membered heterocycleincluding the N and the Cα carbon; R₂ is absent or represents one ormore substitutions to the ring A, each of which can independently be ahalogen, a lower alkyl, a lower alkenyl, a lower alkynyl, a carbonyl(such as a carboxyl, an ester, a formate, or a ketone), a thiocarbonyl(such as a thioester, a thioacetate, or a thioformate), an amino, anacylamino, an amido, a cyano, a nitro, an azido, a sulfate, a sulfonate,a sulfonamido, —(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇; R₃ represents hydrogen or a halogen, a loweralkyl, a lower alkenyl, a lower alkynyl, a carbonyl (such as a carboxyl,an ester, a formate, or a ketone), a thiocarbonyl (such as a thioester,a thioacetate, or a thioformate), an amino, an acylamino, an amido, acyano, a nitro, an azido, a sulfate, a sulfonate, a sulfonamido,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₇; R₅ represents H, an alkyl, an alkenyl, analkynyl, —C(X₁)(X₂)X₃, —(CH₂)m—R₇, —(CH₂)n—OH, —(CH₂)n—O-alkyl,—(CH₂)n—O-alkenyl, —(CH₂)n—O-alkynyl, —(CH₂)n—O—(CH₂)_(m)—R₇,—(CH₂)n—SH, —(CH₂)n—S-alkyl, —(CH₂)n—S-alkenyl, —(CH₂)n—S-alkynyl,—(CH₂)n—S—(CH₂)m—R₇, —C(O)C(O)NH₂, —C(O)C(O)OR′₇; R₆ representshydrogen, a halogen, a alkyl, a alkenyl, a alkynyl, an aryl,—(CH₂)_(m)—R₇, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-alkyl, —(CH₂)_(m)—O-alkenyl,—(CH₂)_(m)—O-alkynyl, —(CH₂)_(m)—O—(CH₂)_(m)—R₇, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-alkyl, —(CH₂)_(m)—S-alkenyl, —(CH₂)_(m)—S-alkynyl,—(CH₂)_(m)—S—(CH₂)_(m)—R-₇, R₇ represents, for each occurrence, asubstituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, orheterocycle; R₃₀ represents a C-terminally linked amino acid residue oramino acid analog, or a C-terminally linked peptide or peptide analog,or an amino-protecting group, or

R₃₂ and R₆₁, independently, represent small hydrophobic groups,preferably lower alkyls, and more preferably methyl; Y₁, and Y₂ canindependently or together be OH, or a group capable of being hydrolyzedto a hydroxyl group, including cyclic derivatives where Y₁ and Y₂ areconnected via a ring having from 5 to 8 atoms in the ring structure(such as pinacol or the like), m is zero or an integer in the range of 1to 8; and n is an integer in the range of 1 to
 8. 33. The method ofclaim 32, wherein administering the boronyl peptidomimetic reduces oneor more of insulin resistance, glucose intolerance, hyperglycemia,hyperinsulinemia, obesity, hyperlipidemia, hyperlipoproteinemia.
 34. Themethod of claim 32, wherein the boronyl peptidomimetic has an EC50 formodification of glucose metabolism which is at least one order ofmagnitude less than its EC50 for immunosuppression.
 35. The method ofclaim 32, wherein the boronyl peptidomimetic has an EC50 for inhibitionof glucose tolerance in the nanomolar or less range
 36. The method ofclaim 32, wherein the boronyl peptidomimetic has an EC50 forimmunosuppression in the μM or greater range.
 37. The method of claim32, wherein the boronyl peptidomimetic is orally active.
 38. A methodfor modifying glucose metabolism of an animal, comprising administeringto the animal a composition including boronyl inhibitor ofpeptidomimetic of a peptide selected from the group consisting Pro-Pro,Ala-Pro, and (D)-Ala-(L)-Ala.